Viral protein R regulates nuclear import of the HIV-1 pre-integration complex.
Replication of human immunodeficiency virus type 1 (HIV-1) in non-dividing cells critically depends on import of the viral pre-integration complex into the nucleus. Genetic evidence suggests that viral protein R (Vpr) and matrix antigen (MA) are directly involved in the import process. An in vitro assay that reconstitutes nuclear import of HIV-1 pre-integration complexes in digitonin-permeabilized cells was used to demonstrate that Vpr is the key regulator of the viral nuclear import process. Mutant HIV-1 pre-integration complexes that lack Vpr failed to be imported in vitro, whereas mutants that lack a functional MA nuclear localization sequence (NLS) were only partially defective. Strikingly, the import defect of the Vpr- mutant was rescued when recombinant Vpr was re-added. In addition, import of Vpr- virus was rescued by adding the cytosol of HeLa cells, where HIV-1 replication had been shown to be Vpr-independent. In a solution binding assay, Vpr associated with karyopherin alpha, a cellular receptor for NLSs. This association increased the affinity of karyopherin alpha for basic-type NLSs, including that of MA, thus explaining the positive effect of Vpr on nuclear import of the HIV-1 pre-integration complex and BSA-NLS conjugates. These results identify the biochemical mechanism of Vpr function in transport of the viral pre-integration complex to, and across, the nuclear membrane.
Popov
S
S
The Picower Institute for Medical Research, Manhasset, NY 11030, USA.
Rexach
M
M
Zybarth
G
G
Reiling
N
N
Lee
M A
MA
Ratner
L
L
Lane
C M
CM
Moore
M S
MS
Blobel
G
G
Bukrinsky
M
M
D001692:Biological Transport / Q000187:drug effects / Q000235:genetics; D002463:Cell Membrane Permeability; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism* / Q000821:virology*; D002474:Cell-Free System / Q000502:physiology; D003593:Cytoplasm / Q000502:physiology; D003673:Defective Viruses / Q000235:genetics / Q000378:metabolism; D004072:Digitonin / Q000494:pharmacology; D015683:Gene Products, gag / Q000502:physiology; D016336:Gene Products, vpr / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D015488:HIV Antigens / Q000502:physiology; D015497:HIV-1 / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D009687:Nuclear Proteins / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D014764:Viral Proteins; D016662:Virus Integration; D028901:alpha Karyopherins; D054301:gag Gene Products, Human Immunodeficiency Virus; D054325:vpr Gene Products, Human Immunodeficiency Virus
D015683:Gene Products, gag; D016336:Gene Products, vpr; D015488:HIV Antigens; D009687:Nuclear Proteins; D014764:Viral Proteins; D028901:alpha Karyopherins; D054301:gag Gene Products, Human Immunodeficiency Virus; C054858:p17 protein, Human Immunodeficiency Virus Type 1; D054325:vpr Gene Products, Human Immunodeficiency Virus; D004072:Digitonin